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The role of FMT and FIS1A in mitochondrial morphology and salt stress in Arabidopsis thaliana

Inaugural-Dissertation

zur

Erlangung des Doktorgrades

der Mathematisch-Naturwissenschaftlichen Fakultät der Universität zu Köln

vorgelegt von Alexandra Ralevski

Köln, October 2016

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Berichterstatter: Prof. Dr. Jens Brüning

Prof. Dr. Thomas Langer

Prof. Dr. Tamas Horvath

Prüfungsvorsitzender: Prof. Dr. Peter Kloppenburg

Tag der mündlichen Prüfung: 28 Oct 2016

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Table of Contents

Abbreviations ... III   Nomenclature ... III   List of figures ... IV   Abstract ... V   Zusammenfassung ... VI  

1. Introduction ... 1  

1.1 Effects of soil salinity on agriculture ... 1  

1.2 Effects of soil salinity on plants ... 2  

1.3 Plant responses to salinity ... 3  

1.4 Mitochondrial response to stress ... 4  

1.4.1 The hypersensitive response and programmed cell death ... 4  

1.4.2 The SOS Pathway ... 5  

1.4.3 Reorganization of Root System Architecture and salt-avoidance tropism ... 6  

1.5 Mitochondrial clustering in response to stress ... 8  

1.6 The FMT/CLU and FIS1A genes in eukaryotes ... 9  

1.7 Thesis aims ... 12  

2. Material and Methods ... 13  

2.1 Material ... 13  

2.1.1. Antibiotics ... 13  

2.1.2 Bacterial strains ... 13  

2.1.3 Primers for PCR-based amplification methods ... 13  

2.1.4 Cloning vectors ... 14  

2.1.5 Plant lines ... 15  

2.1.6 Media, buffers, solutions ... 15  

2.2 Methods ... 18  

2.2.1 Plant growth conditions and seed sterilization ... 18  

2.2.2 Genomic DNA extraction from plant material ... 18  

2.2.3 PCR reaction ... 19  

2.2.4 Mutant screen ... 20  

2.2.5 pFASTG02-FMT reporter construct ... 20  

2.2.6 Sequencing ... 21  

2.2.7 Agrobacterium-mediated transformation of Arabidopsis thaliana ... 21  

2.2.8 Screening and identification of transgenic Arabidopsis seed ... 22  

2.2.9 Phenotypic quantification and statistical analysis ... 22  

2.2.10 Transmission electron microscopy ... 23  

2.2.11 Quantification and analysis of Arabidopsis root cells from transmission electron microscopy ... 23  

3. Results ... 26  

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3.2 Overexpression of the FMT gene in Arabidopsis thaliana ... 28  

3.3 Phenotypic analyses of the fis1A, fmt, and FMT-OE mutants ... 29  

3.3.1 fis1A mutants have shorter roots and slightly shorter leaves ... 29  

3.3.2 fmt mutants have shorter roots and leaves and take longer to flower ... 30  

3.3.3 FMT-OE mutants have a lower rate of germination, take longer to germinate, and have shorter roots and leaves ... 31  

3.4 Effects of salt stress on fis1A, fmt, and FMT-OE mutants and WT plants ... 34  

3.5 Electron microscopy analysis of WT, fis1A, fmt, and FMT-OE plants ... 48  

3.5.1 Salt stress affects various mitochondrial parameters in WT, fis1A, and fmt .... 51  

3.5.2 Clustering is regulated by FMT and is sensitive to salt stress ... 57  

4. Discussion ... 60  

4.1 FMT regulates multiple cellular processes in a variety of organisms ... 60  

4.2 fmt and FMT-OE mutants show deficits in root and leaf length, and delays in flowering and germination ... 63  

4.2.1 Putative role of FMT in germination and flowering control in Arabidopsis ... 64  

4.3 Changes in FMT expression affect mitochondrial size, number, and clustering in columella cells ... 65  

4.4 fis1A mutants have shorter roots and may regulate mitochondrial clustering ... 66  

4.5 Salt stress affects various phenotypic and mitochondrial parameters ... 68  

4.6 Conclusion and future perspectives ... 70  

References ... 72  

Danksagung ... 82  

Erklärung ... 83  

Lebenslauf ... 84  

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Abbreviations

°C degree Celsius

% percent

cDNA complementary DNA

Col-0 Columbia-0

DNA deoxyribonucleic acid

dNTP deoxynucleoside triphosphate E. coli Escherichia coli

e.g. exempli gratia (Latin) for example et al. et alterni (Latin) and others

EtOH ethanol

Fig. figure

hr hour

g gram

GFP green fluorescent protein

kb kilo base pair

L liter

M Molar

min minute

ml mililiter

n number

P value Probability value

PCR polymerase chain reaction rpm revolutions per minute

Sec second

T-DNA transfer DNA

WT wild type

µl microliter

Nomenclature

The wild type genotype is written in italicized capital letters (e.g. FMT).

The mutant genotype is written in italicized lower case letters (e.g. fmt).

The polypeptide products of genes are written in non-italicized, capital letters (e.g. FMT).

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List of figures

Figure 1. Mechanistic action of tropic growth in the Arabidopsis thaliana root.…...8 Figure 2. Salt-regulated spatio-temporal expression in the Arabidopsis root……...28 Figure 3. Seeds of Arabidopsis plants transformed with pFASTG02-FMT give green

fluorescence………..………….29 Figure 4. Phenotypic differences between WT, fis1A, fmt, and FMT-OE lines under

control conditions……….…...32 Figure 5. Visualization of phenotypic differences between WT, fis1A, fmt, and

FMT-OE lines under control conditions.………..……...…...33 Figure 6. Salt stress affects days to germination and flowering, as well as leaf and

root length in wild type Arabidopsis………...……..…35 Figure 7. Salt stress affects days to germination and flowering, as well as leaf and

root length in fis1A mutants………...…37 Figure 8. Days to flowering is increased in fis1A mutants compared to the WT under

salt-stressed conditions.…….………..…..39 Figure 9. Salt stress affects days to germination in fmt mutants………...41 Figure 10. Days to flowering is increased and root and leaf length are decreased in

fmt mutants compared to the WT under salt-stressed conditions……...43 Figure 11. Salt stress affects percent germination, days to germination, and leaf and

root length in FMT-OE mutants...………...45 Figure 12. Germination percentage and root length are decreased in FMT-OE mutants

compared to the WT under salt-stressed conditions.…………...……...47 Figure 13. Differences in mitochondria between WT, fis1A, fmt, and FMT-OE lines

under control conditions.………...………...….50 Figure 14. Comparison of various mitochondrial parameters between WT, fis1A, fmt,

and FMT-OE lines.………....51 Figure 15. Differences in mitochondria between WT, fis1A, and fmt lines under salt

stress………..…….53 Figure 16. Visualization of the effects of salt stress on WT, fis1A, and fmt lines...54 Figure 17. Comparison of various mitochondrial parameters between WT plants

under control and salt-stressed conditions……….………....55 Figure 18. Comparison of various mitochondrial parameters between fis1A mutants

under control and salt-stressed conditions………..……..….56 Figure 19. Comparison of various mitochondrial parameters between fmt mutants

under control and salt-stressed conditions………..……..….57 Figure 20. Clustering patterns in WT, fis1A, fmt, and FMT-OE lines under control

and salt-stressed conditions.………....…..59

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Abstract

Salt stress is known to have severe effects on plant health and fecundity, and mitochondria are known to be an essential part of the plant salt stress response.

Arabidopsis thaliana serves as an excellent model to study the effects of salt stress as

well as mitochondrial morphology. Arabidopsis contains several homologues to known

mitochondrial proteins, including the fission protein FIS1A, and FMT, a homologue of

the CLU subfamily. We sought to examine the effects of salt stress on knockout lines of

FIS1A and FMT, as well as a transgenic line overexpressing FMT (FMT-OE) in

columella cells in the root cap of Arabidopsis. fmt mutants displayed defects in both root

and leaf growth, as well as a delay in flowering time. These mutants also showed a

pronounced increase in mitochondrial clustering and number. FMT-OE mutants

displayed severe defects in germination, including a decrease in total germination, and an

increase in the number of days to germination. fis1A mutants exhibited shorter roots and

slightly shorter leaves, as well as a tendency towards random mitochondrial clustering in

root cells. Salt stress was shown to affect various mitochondrial parameters, including an

increase in mitochondrial number and clustering, as well as a decrease in mitochondrial

area. These results reveal a previously unknown role for FMT in germination and

flowering in Arabidopsis, as well as insight into the effects of salt stress on mitochondrial

morphology. FMT, along with FIS1A, may also help to regulate mitochondrial number

and clustering, as well as root and leaf growth, under both control and salt-stressed

conditions. This has implications for both FMT and FIS1A in whole-plant morphology

as well as the plant salt stress response.

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Zusammenfassung

Salzstress hat schwerwiegende Auswirkungen für die Gesundheit und Fruchtbarkeit von Pflanzen, und Mitochondrien sind ein wesentlicher Teil der Salzstressantwort. Die Arabidopsis thaliana dient als ein hervorragendes Modell, um die Auswirkungen von Salzstress sowie mitochondriale Morphologie zu studieren. Arabidopsis enthält mehrere Homologe zu bekannten mitochondrialen Proteinen, einschließlich des Spaltungsproteins FIS1A, und FMT, ein Homolog des CLU Unterfamilie. Das Ziel war es, die

Auswirkungen von Salzstress auf die Knockout-Linien FIS1A und FMT sowie eine transgene Linie überexprimierenden FMT (FMT-OE) in Columella-Zellen in der Wurzelkappe von Arabidopsis zu untersuchen. Fmt-Mutanten zeigten Defekte im Wurzel- und Blattwachstum, sowie eine Verzögerung in der Blütezeit. Diese Mutanten zeigten auch eine deutliche Zunahme der mitochondrialen Cluster-Bildung und Anzahl.

FMT-OE-Mutanten zeigten schwere Defekte in der Keimung, einschließlich einer

Verringerung der Gesamtkeime und eine Zunahme in der Anzahl der Tage zur Keimung.

fis1A-Mutanten zeigten kürzere Wurzeln und etwas kürzere Blätter, sowie eine Tendenz zur zufälligen mitochondrialen Clustering in Wurzelzellen. Salzstress hatte Einfluss auf verschiedene mitochondriale Parameter, einschließlich einer Zunahme der

Mitochondrienzahl und -gruppierung, sowie eine Abnahme des mitochondrialen

Bereichs. Diese Ergebnisse zeigen eine bisher unbekannte Rolle für FMT in Keimung

und Blüte in Arabidopsis, sowie einen Einblick in die Auswirkungen von Salzstress auf

die mitochondriale Morphologie. FMT, zusammen mit FIS1A, kann auch helfen,

mitochondriale Anzahl und Cluster-Bildung sowie Wurzel- und Blattwachstum zu

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regulieren, sowohl unter Kontroll- und Salzstressbedingungen. Dies hat Auswirkungen

auf beide FMT und FIS1A in Ganzpflanzenmorphologie und für die Salzstressantwort.

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1. Introduction

The driving force behind agriculture is an ever-increasing demand to grow food to sustain a rapidly increasing global population. The pressure to continually produce more crops in a wider variety of environments further drives the need for scientists to design and develop more stress-resistant crop plants. Crops that can endure the effects of exposure to multiple abiotic or biotic stresses while maintaining fecundity will prove to be the most useful for farmers operating in the growing global agricultural market.

1.1 Effects of soil salinity on agriculture

The effects of stress on the proper development and growth of crops currently poses a

severe threat to agriculture. One of the most damaging stresses is salinity, or increased

levels of salt in the soil. All soil contains some level of salt, and many salts, such as

nitrates, are essential plant nutrients. However, the increased use of irrigation and

brackish water, as well as increased runoff and poor drainage, has led to high levels of

excess salt in the soil. Additional sources of excess salt include inorganic fertilizers,

manure, compost, mineral weathering, seawater intrusion into aquifers, and ice melters

used on sidewalks and roads (Hasegawa et al., 2000, Carillo et al., 2011). In addition to

sodium (Na

+

), irrigation waters may also contain higher levels calcium (Ca

2+

) and

magnesium (Mg

2+

). However, when the water evaporates, the Ca

2+

and Mg

2+

precipitate

into carbonates, leaving behind high levels of Na

+

. Soil is considered saline when

solution extracted from the soil has an electrical conductivity of 4dS m

-1

(decisiemens per

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spatially and seasonally, and additional factors such as temperature, pressure, and humidity, can also affect salt levels (Cardon, 2007). It has been estimated that more than 45 million hectares of crops had been damaged by salinization, and 1.5 million hectares were deemed unusable each year (Munns & Tester, 2008). This is predicted to result in up to a 50% reduction in arable land by the year 2050 (Pitman & Läuchli, 2002).

1.2 Effects of soil salinity on plants

Plants can be divided into two major categories for coping with salt tolerance.

Glycophytes (salt-intolerant plants) evolved under conditions of low soil salinity and cannot grow or are severely inhibited at salt concentrations above 150mM NaCl. Most glycophytes can tolerate salt concentrations of ~50mM NaCl and below, although some can survive at higher concentrations. Halophytes (salt-tolerant plants) evolved in places with highly salinized soil, and can survive salinity in excess of 300-400mM (Hasegawa et al., 2000).

High salinity affects plants in two main ways: osmotic stress reduces the ability of the

plant to extract water from the soil, and high concentrations of salts within the plant can

cause damage to plant structures and impede many physiological and biochemical

processes. Initial exposure to salt stress has an immediate effect on the plant, rapidly

increasing the levels of osmotic and ionic stress. Osmotic stress occurs as a result of

excess Na

+

ions in the surrounding soil compared to internal concentrations in the root,

which generates an external osmotic pressure that reduces water influx into the root. The

result is a water deficit similar to those seen under drought conditions. This can lead to

impaired growth and decreased viability, as water and key nutrients are unable to be

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transported throughout the plant. Osmotic stress is believed to occur for the duration of salt exposure, resulting in increased stomatal closure and an inhibition of cell division and expansion. Long-term exposure to salt stress can also trigger ionic stress, which occurs as a result of increased Na

+

accumulation in the leaves, which can disrupt protein synthesis and enzymatic activity, often triggering premature senescence in older leaves.

This reduces the photosynthetic availability of the plant, further impairing plant growth (Hasegawa et al., 2000, Carillo et al., 2011). However, despite the damaging effects of salinity, plants have evolved a variety of mechanisms to counter-act salt stress over both the short- and long-term.

1.3 Plant responses to salinity

Plants display a wide variety of responses to salinity, and as a result exhibit several

whole-organism phenotypic changes. Some of these changes are side effects, while some

occur as a direct response to salt stress. One example of a side-effect-derived change

occurs when excess Na

+

ions are actively shuttled out of the plant shoot and into the

leaves, in order to allow for K

+

accumulation in the shoot to help balance the K

+

/Na

+

ion

ratio. However, when Na

+

ions reach a critical level in the leaves they begin to stunt

growth, eventually leading to leaf necrosis and eventual plant death (Hauser & Horie,

2010). Direct changes in response to salt stress include the reorganization of root system

architecture (RSA), which allows for a rapid response to changes in NaCl in the soil

(Malekpoor Mansoorkhani et al., 2014, Jones & Ljung, 2012). Other direct responses

include a suppression of germination or a delay in flowering, most likely as a way of

waiting until conditions are more ideal to grow or produce offspring seeds (Srivastava et

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al., 2016, Conti et al., 2008). The genes and pathways that control these phenotypic changes are known in some cases, but the overall etiology underlying the various salt stress responses remain elusive. In addition, the roles of various organelles, including mitochondria, which are known to play a role in the stress response, remain poorly understood. Below is a discussion of some of the most well understood plant responses to stress, including salinity, and the role of mitochondria in each of these responses.

1.4 Mitochondrial response to stress

Mitochondria are known to play key roles in a variety of plant responses to salt stress.

These responses usually involve highly conserved mechanisms, and include the

hypersensitive response (HR) and programmed cell death (PCD), the SOS Pathway, the reorganization of Root System Architecture and salt-avoidance tropism.

1.4.1 The hypersensitive response and programmed cell death

A common response to stress is the plant hypersensitive response (HR). In response to

pathogenic attack, cells will undergo programmed cell death (PCD) in the surrounding

area under attack, disabling a virus from co-opting host machinery from neighbouring

cells, eventually rendering the virus unable to reproduce, and thus eventually die. PCD is

also a well-characterized mechanism in animals, and many of the basic regulatory

mechanisms that underlie this response are similar in both plants and animals. Indeed,

short-term salinity stress was shown to induce PCD in a manner similarly to animals

(Andronis & Roubelakis-Angelakis, 2010). One shared feature between animal and plant

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mitochondria may initiate apoptosis in response to changes in various cellular regulators, such as cytosolic calcium and cellular pH, or indicators of cellular energy availability, such as ATP, ADP, NADH, and NADPH. Various other proteins may also be activated in response to stress and can associate with and modify the permeability of the outer mitochondrial membrane (OMM), including the opening of the mitochondrial permeability transition pore (mPTP). This leads to a decrease in the mitochondrial membrane potential and the release of various cell death activators from within the mitochondrion, including the apoptosis-inducing factor (AIF) and cytochrome c (Lam et al., 2001, Morel & Dangl, 1997, Heath, 2000).

1.4.2 The SOS Pathway

The SOS (Salt Overly Sensitive) pathway was originally identified in a genetic screen to find plants that were hypersensitive to salt stress (Wu et al., 1996, Zhu et al., 1998). The sos1, sos2, and sos3 mutants were shown to have severely impaired growth on media with an excess of Na

+

or Li

+

ions, or a deficit of K

+

ions, but grew similarly to wild type plants under normal growth conditions. These mutants also grew normally under general osmotic or drought stress, which indicates that the SOS genes play a specific role in mediating the ionic response to salt stress in plants. The SOS pathway is activated when excess Na

+

is sensed by the cell, leading to an increase of cytoplasmic Ca

2+

. This Ca

2+

spike is sensed by SOS3, which activates SOS2, forming a SOS3-SOS2 kinase complex.

This complex activates SOS1, an Na

+

/H

+

antiporter, which pumps excess Na

+

from the

cytoplasm and into extracellular space or the root medium (Ji et al., 2013). Mitochondria

are known to buffer cytosolic calcium following a spike in concentration (Vandecasteele

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et al., 2001), however, the extent of their role in the SOS pathway has not been well studied.

1.4.3 Reorganization of Root System Architecture and salt-avoidance tropism

The shape and structure of the roots, as well as their spatial configuration within the soil, determines the root system architecture (RSA) of a plant. The RSA of an individual plant is determined by the unique and heterogeneous distribution of edaphic resources (Malamy, 2005, de Dorlodot et al., 2007). In response salt stress, the RSA is altered such that primary root elongation is inhibited, while lateral root (LR) formation increases in response to lower concentrations of NaCl, but is inhibited at higher concentrations (Wang et al., 2008, Zolla et al., 2010). These responses are mediated by changes in cell length and number (Duan et al., 2015), and mitochondria are known to play a role in this regulation (van der Merwe et al., 2009).

In addition to changes in RSA, plant roots may change their direction of growth to avoid

excess salt in the soil. Roots primarily grow downwards towards the gravity vector, a

phenomenon known as positive gravitropism, or tropic growth. Although it is not known

exactly how roots recognize the gravity vector, the “starch statolith hypothesis” posits

that amyloplasts in the columella cells of the root cap sediment to the “bottom” of the

cell, directing the orientation of growth (Fig. 1A,B) (Sato et al., 2015). When it is

necessary to avoid NaCl ions in the soil, roots can exhibit negative gravitropism and

grow against the gravity vector, a process known as salt-avoidance tropism, which helps

minimize exposure to stress. How the root is able to sense excess salt and subsequently

change its direction of growth is not well understood. Sun et al. (2008) found that, upon

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exposure of the root to salt stress, two main responses were initiated: 1) rapid degradation of amyloplasts, followed by 2) root bending resulting in negative gravitropism.

Amyloplast degradation may be regulated by the SOS pathway, and root bending is likely triggered by PIN2, an auxin efflux carrier, that asymmetrically distributes auxin in the root leading to root curvature (Ottenschlager et al., 2003). However, it is highly likely that other proteins, including those involved with mitochondria, act to regulate salt- avoidance tropism and root bending, as well as reorganization of RSA. Zhang et al.

(2015) recently discovered a mitochondrial-localized protein, SSR1, which regulates root

growth and architecture, and is required for PIN2 trafficking. This indicates a clear role

for mitochondria and their associated proteins in the regulation of root system

architecture, with implications for a role in the changes of this architecture in response to

salt stress.

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Figure 1: Mechanistic action of tropic growth in the Arabidopsis thaliana root. A) Following a 90°

turn, statoliths in columella cells start to fall to the bottom of the cell, and are fully sedimented by 5 min. B) Diagram of an Arabidopsis root; columella cells are labelled in green (adapted from Barrada et al. (2015);

Sato et al. (2015)). Scale bar = 50 µm.

1.5 Mitochondrial clustering in response to stress

A less well-understood response of mitochondria to stress is that of mitochondrial

clustering. Mitochondrial trafficking and movement is known to be essential for proper

mitochondrial and cellular function, but under stress conditions, mitochondria display

altered motility and distribution, which can have deleterious consequences for an

organism (Chen & Chan, 2009, Nunnari & Suomalainen, 2012). In plants, mitochondrial

clustering and/or arrest of mitochondrial motility has been recognized as a response to

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oxygen species, heat shock (Scott & Logan, 2008), methyl jasmonate (Zhang & Xing, 2008), oxylipin, 9-hydroxy-10,12,15-octadecatrienoic acid (Vellosillo et al., 2013), and UV light exposure (Gao et al., 2008). However, the mechanisms that give rise to this mitochondrial clustering are not known. Knockouts of the highly conserved gene CLU (CLUstered mitochondria) was shown to induce mitochondrial clustering in a variety of eukaryotes, including Dictyostelium, Saccharomyces cerevisiae, Drosophila, and Arabidopsis (Zhu et al., 1997, Fields et al., 1998, Cox & Spradling, 2009, El Zawily et al., 2014). However, the role of mitochondrial clustering in plants in response to stress, including salt stress, has not previously been investigated.

1.6 The FMT/CLU and FIS1A genes in eukaryotes

The first member of the CLU family to be identified was cluA in Dictyostelium, and this gene was found to be necessary for the correct dispersion of mitochondria within the cell (Zhu et al., 1997). Fields et al. (1998) demonstrated that CLU1, a functional homologue of cluA in Saccharomyces cerevisiae (S. cerevisiae), performed a similar function. clu1∆

cells, which had their CLU1 genes deleted, formed loose clusters of mitochondria within the cytoplasm. Cox and Spradling (2009) characterized the CLU gene in Drosophila, known as clueless, and found that clueless mutants exhibited mitochondrial clustering within cells. Flies that were homozygous for the clu defect (clu

d08713

or clu

f04554

) lived for only 3-7 days, were smaller than WT flies, sterile, and could not fly.

The CLU homologue in Arabidopsis, FMT (friendly mitochondria), was originally

identified by Logan et al. (2003) in a mutant screen to find candidate genes involved in

mitochondrial dynamics and morphology in higher plants. FMT is 26% identical and

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41% similar to the Dictyostelium cluA protein and, like cluA, also contains a TPR (tetratricopeptide repeat)-like domain. FMT is 20% identical and 34% similar to the S.

cerevisiae Clu1p protein. All CLU homologues that have been studied posses a TPR domain, and it remains the most highly conserved portion of the CLU gene throughout its evolution between species. Tetratricopeptide repeats are found in genes in all species, and are known for their ability to mediate protein interactions between partner proteins.

In plants, they are found in a variety of genes involved in stress and hormone signalling.

One example is TTL1 (Tetratricopeptide-repeat thioredoxin-like 1), which is known to be a positive regulator of the ABA- (abscisic acid) mediated stress response. Knockouts of TTL1 increased salt and osmotic sensitivity during seed germination and in later development (Rosado et al., 2006). Drosophila clueless was also found to bind nuclear- encoded mitochondrial mRNAs through its TPR domain and direct them to the mitochondrial outer membrane where they could potentially be positioned for co- translational import into mitochondria (Sen et al., 2015).

Electron microscopy analysis of leaf tissue of fmt mutant plants initially revealed the

similar phenotype of mitochondrial clustering that was observed in other species (Logan

et al., 2003). Further analysis by El Zawily et al. (2014) revealed that FMT might play a

role in intermitochondrial association and quality control. It was hypothesized that FMT

may function as a fusion protein, as there are currently no known homologues to

conserved fusion proteins in plants. However, plant fission proteins are highly conserved

in plants, including DRP and FIS1A. Mitochondrial fission proteins also play an

important role in the stress response by facilitating the division of a partially damaged

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mitochondrion into one healthy and one damaged mitochondrion that can be targeted for degradation (Youle & van der Bliek, 2012).

The mitochondrial division machinery used by Arabidopsis is conserved across animals, plants, and fungi (for review see Praefcke & McMahon, 2004). In plants, dynamin-like proteins (DLPs) have been shown to be necessary for the division of mitochondria (Aung

& Hu, 2012). DRP3A and DRP3B (previously known as ADL2a and ADL2b,

respectively) in Arabidopsis are homologous to the Dnm1 and Drp1/DLP1 proteins found

in yeast and mammals, respectively. These proteins are part of a DRP subclade that is

well conserved across eukaryotic species and contain the GTPase, MD, and GED

domains (Miyagishima et al., 2008). DRP3A and DRP3B both localize to mitochondria

and were shown to play a dual role in the final scission of both organelles (Aung & Hu,

2012). Arabidopsis also contains two proteins homologous to FIS1 in S. cerevisiae and

humans: FIS1A (also known as BIGYIN1), and FIS1B (also known as BIGYIN2)

(Mozdy et al., 2000, Tieu & Nunnari, 2000, Smirnova et al., 2001, James et al., 2003,

Youle & Karbowski, 2005). Similar to their yeast and mammalian counterparts, these

plant proteins localize to the outer mitochondrial membrane (OMM) and play a key role

in mitochondrial division (Logan, 2010). Arabidopsis fis1A mutants had a reduction in

the number of mitochondria per cell, with simultaneous increases in the size of individual

mitochondrion in protoplasts and leaves (Scott et al., 2006). This provides further

evidence for the role of FIS1A in mitochondrial fission in plants. However, the role of

fission and FIS1A during salt stress is not currently known.

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1.7 Thesis aims

The plant salt stress response remains an important mechanism for maintaining growth and survival under ever-changing environmental conditions, and mitochondria are known to be essential for the mediation of this response. However, how this organelle exerts its control, and what proteins are involved, is not well understood. Two mitochondrial proteins, FIS1A, and FMT, are to known be essential for mitochondrial quality control.

Given the role of mitochondria in the salt stress response, and given the fact that salt

stress is sensed first in the soil by the roots, we wanted to examine the effects of a

knockout of either FMT or FIS1A, as well as an overexpression of FMT, in mitochondria

in columella cells of the roots under both control and salt-stressed conditions. We also

wanted to examine the phenotypic effects of these mutants under both control and salt-

stressed conditions. Additionally, since the effects of salt stress on mitochondrial

morphology in wild type plants had not previously been characterized, we wanted to

examine various mitochondrial parameters in the columella cells of the roots of wild type

plants exposed to salt stress. The aim is to further our understanding of the role of

mitochondria in salt stress and as such add to the cannon of knowledge of the salt stress

response as a whole.

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2. Material and Methods

2.1 Material

2.1.1. Antibiotics

Table 1: Antibiotics used in this study

Antibiotic Solvent Stock concentration (mg/ml)

Working concentration ( µ g/ml)

Gentamicin H

2

O 10 50

Kanamycin H

2

O 50 50

Spectinomycin H

2

O 100 75

2.1.2 Bacterial strains E. coli

One Shot TOP10 (Invitrogen, USA) DH5α (Invitrogen, USA)

Agrobacterium tumefaciens GV3101 (pMP90)

2.1.3 Primers for PCR-based amplification methods

All primers were purchased from the W.M. Keck Foundation (Yale School of Medicine,

New Haven, CT). Primer sequences are listed in Table 2.

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Table 2: Primers used in this study

Primer Name Sequence (5’  3’) Notes

T-DNA Primers

LBb1.3 ATTTTGCCGATTTCGGAAC Left border primer for T-DNA insertion FIS1A LP AAGATCCTCCTTGACCTCGAC Left primer for FIS1A

(SALK_006512C) FIS1A RP GCTGATTGGAGACAAGCTTTG Right primer for FIS1A

(SALK_006512C) FMT LP ATACCTGCAGCAGTTTGCAAC Left primer for FMT

(SALK_046271C) FMT RP CTAGCGCCAACAGCTCTACTG Right primer for FMT

(SALK_046271C) Gateway Primers

attB1 FP FMT GGGGACAAGTTTGTACAAAAAAGCAG GCTTCATGGCTGGGAAGTCGAAC

attB1 Forward primer for FMT attB1 RP FMT GGGGACCACTTTGTACAAGAAAGCTG

GGTCTTTTTTGGCTTTTTGCTTCTT

attB1 Reverse primer for FMT Sequencing Primers

M13 FP GTAAAACGACGGCCAG Forward sequencing primer for pDONR 221 M13 RP CAGGAAACAGCTATGAC Reverse sequencing

primer for pDONR 221 FMT Seq1 ATGGCTGGGAAGTCGAAC FMT Sequencing

Primer 1 FMT Seq2 ATCTATCAGAGCGCATGTTCA FMT Sequencing

Primer 2 FMT Seq3 GAGCAGAAGAAGCACTTACCA FMT Sequencing

Primer 3 FMT Seq4 GCCATAGGGTTGTTGCTCAG FMT Sequencing

Primer 4 FMT Seq5 AAGAGGAGATAGCTGCTGATG FMT Sequencing

Primer 5 FMT Seq6 TAATCTTTGCCAAAAGGTTGGTG FMT Sequencing

Primer 6 FMT Seq7 AAAATGAGAGACTTCTTGGTCCT FMT Sequencing

Primer 7 FMT Seq8 AACAGAAAACCTGGCTCCTG FMT Sequencing

Primer 8

2.1.4 Cloning vectors

The pDONR 207 (Invitrogen) and pFASTG02 ( p*7FWG2, Plant Systems Biology)

vectors were used for cloning in this study.

(24)

2.1.5 Plant lines

All experiments were performed using Arabidopsis thaliana Col-0 wild type plants or mutants in the Col-0 background. fmt homozygous mutants (SALK_046271C), and fis1A homozygous mutants (SALK_006512C) were obtained from ABRC (Arabidopsis Biological Resource Center, Ohio State University, Columbia, OH, USA). All homozygous mutant lines were confirmed by PCR.

2.1.6 Media, buffers, solutions

Media LB Media

25g LB Broth ddH

2

O to 1L

For solid medium, 2% Agar was added to the above medium.

After autoclaving at 121°C for 20 mins and cooling to 55°C, antibiotics were added.

MS-Agar Media (pH 5.7)

4.3g MS Salts

0.5g MES

10g Agarose

ddH

2

O to 1L

(25)

After autoclaving at 121°C for 20 mins and cooling to 55°C, media was poured into plates.

125mM NaCl Stress Media 800 ml MS-Agar Media 7.3g NaCl

ddH

2

O to 1L

After autoclaving at 121°C for 20 mins and cooling to 55°C, media was poured into plates.

Buffers

CTAB Buffer (100mL, pH 5.0)

2 g CTAB (Hexadecyltrimethylammonium bromide) 10 ml 1 M Tris pH 8.0

4 ml 0.5 M EDTA pH 8.0 28 ml 5 M NaCl

40 ml ddH

2

O

Phosphate Buffer Stock A

27.6g NaH

2

PO

4

·H

2

O

ddH

2

O to 1L

(26)

Phosphate Buffer Stock B 28.4g/L Na

2

HPO

4

·H

2

O ddH

2

O to 1L

0.2M Phosphate Buffer (pH 6.8)

51% Phosphate Buffer Stock A 49% Phosphate Buffer Stock B

Solutions Fixative

0.5% (wt/vol) formaldehyde 3% (wt/vol) gluteraldehyde 0.1M Phosphate Buffer (pH 6.8)

1% osmium tetroxide fixative 1ml OsO

4

(4%)

3ml 0.1M Phosphate Buffer (pH 6.8)

2% uranyl acetate staining solution 0.4g uranyl acetate

20ml H

2

O

(27)

2.2 Methods

2.2.1 Plant growth conditions and seed sterilization

Arabidopsis seeds were sown directly on Fafard #2 soil mixture (Sun Gro Horticulture) and were grown under 16-hr light/8-hr dark (long-day) photoperiods at 22°C +/-1°C under cool-white light at 100 µmol m

-2

s

-1

. For experiments done on sterilized MS (Murashige Skoog)-Agar media, seeds were first surface sterilized by washing in 70%

(v/v) ethanol for 5 sec, and this wash was replaced by 0.1% triton X-100 in 50% bleach (v/v) for 5 sec before five rinses in autoclaved ddH

2

O. Seeds were then plated on 100 x 100 x 15 mm square petri dishes (Ted Pella), and plates were stratified at 4°C for three days in the dark to synchronize germination. Plates were then moved to long-day

photoperiod conditions at 22°C +/-1°C under cool-white light at 100 µmol m

-2

s

-1

. Plates were placed at an angle to allow for root growth along the surface of the agar. For salt- stressed growth conditions on plates, seeds were plated on MS-Agar plates supplemented with 125mM NaCl. For salt-stressed conditions in the soil, the following NaCl

concentrations were added when the plants were watered, beginning one week after germination and increasing every week: 50mM NaCl, 75mM NaCl, 100mM NaCl, 125mM NaCl, 140mM NaCl.

2.2.2 Genomic DNA extraction from plant material

Genomic plant DNA was extracted using the CTAB method. 200 mg plant leaf tissue

was ground in eppendorf tubes and 500µl CTAB Buffer was added. The mixture was

incubated for 15 minutes at 55°C and tubes were then centrifuged at 13,000 rpm for 5

(28)

minutes. The supernatant was transferred to a new eppendorf tube and 250 µl of 24:1 chloroform:isopropanol was added and mixed by inversion. The tubes were spun at 13,000 rpm for 1 minute. The upper aqueous phase was transferred to a new eppendorf tube and 50 µl of 7.5M ammonium acetate and 500 µl of ice-cold absolute ethanol were added. The tubes were mixed slowly by inversion and placed at -20°C for 1 hour to precipitate the DNA. Tubes were then spun at 13,000 rpm for 1 minute to form a pellet.

The supernatant was removed and the pellets were dried for 15 minutes at room temperature. The pellet was resuspended in 50 µl DNase-free H

2

O and stored at 4°C.

2.2.3 PCR reaction

All PCR reactions were done using a Bio Rad C1000 Touch Thermal Cycler. For genotyping, a standard PCR reaction mix was used, using PCR Supermix (Invitrogen).

The standard PCR reaction mix (Table 3) and standard PCR thermal profile (Table 4) are shown below.

Table 3: Standard PCR reaction mix

Reagent Amount Concentration

PCR Supermix 20 µl 1.1X

Forward Primer 0.5 µl 10 µM

Reverse Primer 0.5 µl 10 µM

DNA Template 1 µl 100-150 ng

H

2

O 3 µl N/A

25 µl

(29)

Table 4: Standard PCR thermal profile

Step Temperature Time Cycles

Initial denaturation 95°C 3 min.

Denaturation 95°C 30 sec. 20-35

Annealing 55°C 30 sec. 20-35

Extension 72°C 20-300 sec. 20-35

Final extension 72°C 3 min.

Hold 4°C ∞

2.2.4 Mutant screen

Both FMT and FIS1A were screened for available T-DNA insertion lines on TAIR (The Arabidopsis Information Resource, http://www.arabidopsis.org/). PCR was used to test whether the T-DNA was inserted at the predicted insertion site. All T-DNA insertions were confirmed via PCR using left and right primers flanking the genomic sequence, and a border primer located within the T-DNA sequence (see Table 2 for primer list).

2.2.5 pFASTG02-FMT reporter construct

pFASTG02-FMT was constructed by subcloning a FMT cDNA fragment of the expected size into the pDONR 221 vector (Gateway, Invitrogen). In order to PCR amplify the cDNA fragment, the following primers were used: forward (attB1 FP FMT),

5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCTGGGAAGTCGAAC- 3’, and reverse (attB1 RP FMT),

5’-GGGACCACTTTGTACAAGAAAGCTGGGTCTTTTTTGGCTTTTTGCTTCTT-3’.

The fragment was subsequently cloned into pFASTG02 (Shimada et al., 2010) according

to the manufacturer’s protocol (Invitrogen). This vector construct, pFASTG02-FMT,

(30)

virus (CaMV) 35S promoter . The construct was sequenced to identify an error-free clone and subsequently transformed into wild type Col-0 plants by means of Agrobacterium- mediated transformation using the Agrobacterium tumefaciens strain GV3101 (pMP90).

2.2.6 Sequencing

All sequencing reactions were done by the W.M. Keck Foundation (Yale School of Medicine, New Haven, CT).

2.2.7 Agrobacterium-mediated transformation of Arabidopsis thaliana

The pFASTG02-FMT vector was transformed into the Agrobacterium tumefaciens strain

GV3101 via electroporation and colonies were selected on LB media plates

supplemented with 50 µg/ml spectinomycin. Single colonies were picked and cultured in

LB media supplemented with 50 µg/ml spectinomycin and grown to OD

600

= 0.6. The

cultures were then centrifuged at 13,000 rpm for 30 minutes and the pellets were

resuspended in a 5% sucrose solution. Plants were dipped according to Clough and Bent

(1998) with the following modifications: Silwet L-77 was added to the sucrose solution

to a concentration of 0.05% and Arabidopsis plants with emerging flower stems were

dipped in the solution for five seconds. The plants were then kept under long-day

photoperiod conditions under transparent covers at 22°C +/- 1°C under cool-white light at

100 µmol m

-2

s

-1

for three days. Covers were removed and plants were grown until seed

was mature. Mature seeds were collected and screened to identify transgenic seed

expressing the pFASTG02-FMT vector.

(31)

2.2.8 Screening and identification of transgenic Arabidopsis seed

Seeds from transformed plants were collected and screened for transgenic individuals containing the pFASTG02-FMT vector by the expression of green fluorescence in the seed coat by fluorescence microscopy under 4X magnification with the Zeiss Axioplan 2 fluorescence microscope (Carl-Zeiss, Germany). Transgenic seeds were then sown to produce T

2

seeds. Lines with a single transgene insertion were identified by an ~3:1 segregation ratio of GFP to no-GFP, respectively. Seeds from this line were sown to identify a homozygous plant (FMT-3), which was identified by T

3

seed that exhibited 100% GFP fluorescence. Seeds from line FMT-3 were collected and used in subsequent experiments.

2.2.9 Phenotypic quantification and statistical analysis

Arabidopsis plants were grown to three weeks old in the soil under control or salt- stressed conditions described above. For leaf length quantification, three leaves were selected and measured from the base of each leaf to the tip using a ruler. For quantification of root length under control conditions, Arabidopsis plants were grown on control MS-Agar plates and roots were measured using a ruler at days seven and fourteen.

For quantification of root length under salt-stressed conditions, Arabidopsis plants were grown on control MS-Agar plates for one week, and then transplanted to either control or 125mM NaCl MS-Agar plates for one week and roots were measured at day fourteen.

For MS-Agar plate experiments, at least 20 replicates were used for each experiment, and

each experiment was repeated three times. For soil experiments, at least 10 replicates

were used for each experiment, and each experiment was repeated three times. Mutant

(32)

genotypes were compared to the wild type under both control and salt-stressed conditions.

Statistical differences were determined using Student’s two tailed t test, one-way analysis of variance (ANOVA), or two-way ANOVA, where appropriate.

2.2.10 Transmission electron microscopy

Arabidopsis seedlings were grown on MS-Agar plates for one week, and were then transferred to either 125mM NaCl MS-Agar plates or MS-Agar plates without the addition of NaCl for an additional week. Fixation and embedding of 14-day-old root samples was done according to Wu et al. (2012) with the following modifications:

Durcupan epoxy resin (Sigma) was used for infiltration, tissue was collected on single slot copper grids (EMS) coated with formvar, and no post-sectioning heavy metal staining was used. Transverse sections were cut ~30 µM deep into the columella of the root and subsequently viewed under a Tecnai 12 Transmission Electron Microscope (FEI, USA). At least ten cells from four biological samples each of WT, fis1A, fmt, and FMT- OE roots were examined for control experiments, and at least ten cells from two biological samples each of WT, fis1A, or fmt roots were examined for salt-stressed experiments. Due to the difficulty in preserving salt-stressed FMT-OE mutants during the fixation and embedding process, these mutants were not observed for TEM.

2.2.11 Quantification and analysis of Arabidopsis root cells from transmission electron microscopy

Using Fiji, an individual cell, nucleus, vacuole, and mitochondria were traced and

(33)

mitochondria per cell, and centroid XY coordinates of each individual mitochondrion.

Mitochondrial coverage was calculated as a percent using the following formula:

((Cytoplasmic area–mitochondrial area)*100), where cytoplasmic area = (Cell area–

nuclear area–vacuole area), and mitochondrial area is the sum of all the areas of the individual mitochondria within the cell.

Mitochondrial clustering was calculated using the Nearest Neighbor Distances (NND) tool in the BioVoxxel toolbox plugin in Fiji (http://imagej.net/BioVoxxel_Toolbox). The NND tool measures the average nearest neighbor ratio (ANN), which is calculated as the distance from the center of a particular particle (in this case a mitochondrion) to the center of its nearest neighbor. The average of all the nearest neighbor distances are then taken. If the average distance is less than the average of a hypothetical random distribution, the mitochondria are considered clustered. If the average distance is greater than a hypothetical random distribution, the mitochondria are considered dispersed. The average nearest neighbor ratio (ANN) is given as:

!""  = !

!

!

!

where !

!

is the observed mean distance between each feature and its nearest neighbour:

! !  =

!!!!

! !

!

and !

!

is the expected mean distance for the features given in a random pattern:

! ! = !.!

!/!

(34)

In the above equations, !

!

equals the distance between feature ! and its nearest neighboring feature, ! corresponds to the total number of features, and ! is the area of a minimum enclosing rectangle around all features.

The average nearest neighbor z-score for the statistic is calculated as:

! = !

!

!" !!

!

where:

!" = !.!"#$"

!/!

If the ANN is less than 1, then the pattern exhibits clustering. If the ANN is greater than

1, the pattern trends towards dispersion. If the ANN is exactly 1, the pattern is

considered to be random (Clark & Evans, 1954, Mitchell, 2005). Mitochondria were first

analyzed using the Analyze Particles command in Fiji to analyze and measure the

individual mitochondria of a single cell. The NND plugin was then used to calculate the

ANN of each individual mitochondrion.

(35)

3. Results

Arabidopsis plants lacking a functional FMT gene show severe defects in mitochondrial distribution and movement, as well as deficits in root growth (El Zawily et al., 2014).

Plants lacking a functional FIS1A gene have a reduction in the number of mitochondria per cell, as well as an increase in the size of individual mitochondria in protoplasts and leaves (Scott et al., 2006). How exactly FMT and FIS1A mediate these changes in mitochondria is still unclear. Given the role of mitochondria in the salt stress response, and given the fact that salt stress is sensed first in the soil by the roots, we wanted to examine the effects of a knockout of either FMT or FIS1A, as well as an overexpression of FMT, in mitochondria in the roots under both control and salt-stressed conditions.

Additionally, since the effects of salt stress on mitochondrial morphology in wild type plants has not previously been characterized, we wanted to examine various mitochondrial parameters in the columella cells of the roots of wild type plants exposed to salt stress. These findings will further our understanding of the roles of FMT and FIS1A in mitochondrial morphology, as well as their role(s) in the salt stress response. In addition, it will provide insight into the effects of salt stress on mitochondrial morphology in wild type plants.

3.1 Identification of the FMT and FIS1A genes

The FMT gene was originally identified by Logan et al. (2003) and fmt mutants in

Arabidopsis were shown to have an increased number of clustered mitochondria in the

leaves. These mutants were further characterized by El Zawily et al. (2014), and were

(36)

found to have shorter roots, as well an increase in the association time between mitochondria, as well as an increase in mitochondrial matrix mixing. The FIS1A gene was originally characterized by Scott et al. (2006), and fis1A mutants in Arabidopsis were found to have a reduced number of mitochondria per cell, but an increase in the size of individual mitochondria in protoplasts and leaves. However, the role of these genes with regards to whole-plant morphology, as well as their role in salt stress, has yet to be explored.

3.1.1 Expression levels of FMT and FIS1A in response to salt stress

Expression levels of FMT and FIS1A under salt stress in the Arabidopsis root were examined using the Arabidopsis Spatio-Temporal Root Stress eFP Browser (http://dinnenylab.info/browser/query). This browser examines the expression levels of

~5 day old seedlings exposed to 140mM NaCl from 1 to 48 hours, compared to exposure

on MS-Agar for 1 and 48 hours. A comparison of the expression levels of FIS1A and

FMT to expression levels of the Salt Overly Sensitive genes (SOS1, SOS2, and SOS3),

which are known to be induced by salt stress, is shown in Figure 2. It is clear that FMT

gene expression increases sharply in as little as three hours in the epidermis, with a

moderate increase in expression in the stele and cortex from 1-48 hours following NaCl

exposure. FIS1A is only mildly upregulated in the epidermis in response to salt stress,

similar to SOS1 expression. SOS2 has a moderate decrease in expression in the

epidermis, stele, and cortex, while SOS3 is initially highly upregulated from 1-3 hours,

with an eventual decrease in expression after 48 hours. While this eFP Browser is

informative for short-term exposure at 140mM NaCl, it does not provide information for

(37)

long-term exposure to NaCl stress at different concentrations. An in-depth phenotypic and functional analysis of both FMT and FIS1A under different salt-stressed conditions is therefore essential in furthering our understanding of the role of these genes during salt stress.

Figure 2: Salt-regulated spatio-temporal expression in the Arabidopsis root. A) FIS1A (left) and FMT (right). B) (left to right) SOS1, SOS2, SOS3.

3.2 Overexpression of the FMT gene in Arabidopsis thaliana

In order to further our understanding of the role of FMT in whole-plant and mitochondrial morphology, we created a transgenic Arabidopsis plant line (FMT-OE) overexpressing FMT under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Arabidopsis plants were transformed with Agrobacterium containing the pFASTG02-FMT overexpression vector. The pFASTG02 vector carries a screenable marker that produces a GFP signal visible in the mature seed coat of transformed plants.

Transgenic seeds from these plants were then sown to obtain T

2

seeds. Lines with a

single transgene insertion were identified by an ~3:1 segregation ratio of GFP to no-GFP,

respectively. Transgenic GFP seeds from these lines were sown to identify a

(38)

homozygous plant (FMT-3), which was identified by T

3

seed that exhibited 100% GFP fluorescence compared to WT seed (Fig. 3). Seeds from line FMT-3 were collected and used in subsequent experiments.

Figure 3: Seeds of Arabidopsis plants transformed with pFASTG02-FMT give green fluorescence.

A) (right) GFP-expressing T

3

generation seeds obtained from FMT-3, a homozygous Arabidopsis plant overexpressing the FMT gene in the pFASTG02 vector, (left) non-transformed WT seeds do not give green fluorescence. B) The same field view as in (A), but viewed under bright field light. Scale bar = 100 µm.

3.3 Phenotypic analyses of the fis1A, fmt, and FMT-OE mutants

3.3.1 fis1A mutants have shorter roots and slightly shorter leaves

As described above, a homozygous T-DNA insertion line for FIS1A (SALK_006512C) was found within the SALK collection. When using primers spanning the insertion site, no transcript could be detected via PCR. The T-DNA insertion hypothetically leads to a block of transcription, rendering a truncated or non-functional FIS1A protein.

Under control conditions, fis1A mutant plants did not have a significantly different

germination rate compared to WT plants (98.6% ±3.26% versus 99.2% ±1.88%,

respectively) (Fig. 4A), nor did they take longer to germinate than WT plants (1.5 days

(39)

±0.51 days versus 1.24 days ±0.43 days, respectively) (Fig. 4B). Leaf length was not significantly (p=0.0775) shorter in fis1A mutants compared to the WT (1.03 cm ±0.21 cm versus 1.23 ±0.25 cm, respectively) (Figs. 4C, 5N). These mutants also did not display differences in days to flowering (29.4 days ±1.99 days) compared to the WT (28.89 days

± 1.28 days) (Fig. 4D). However, fis1A mutants did display significantly shorter roots at both 7 (0.59 cm ± 0.17 cm) and 14 (0.85 cm ± 0.43 cm) days old under control conditions compared to the WT (0.86 cm ±0.27 cm and 1.56 cm ±0.48 cm, respectively) (Figs. 4E,F, 5B,F).

3.3.2 fmt mutants have shorter roots and leaves and take longer to flower

As described above, a homozygous T-DNA insertion line for FMT (SALK_046271C) was found within the SALK collection. When using primers spanning the insertion site, no transcript could be detected via PCR. The T-DNA insertion hypothetically leads to a block of transcription, rendering a truncated or non-functional FMT protein.

Under control conditions, fmt mutant plants did not have a significantly different

germination rate (98.6% ±3.13%) (Fig. 4A) nor did they take longer to germinate (1.33

days ±0.48 days) compared to WT plants (Fig. 4B). However, leaf length was

significantly shorter in fmt mutants (0.93 cm ±0.20 cm) (Fig. 4C, 5O), and these mutants

also took significantly longer to flower (33.57 days ±2.82 days) compared to the WT

(Fig. 4D). fmt mutants also displayed significantly shorter roots at both 7 (0.54 cm ± 0.18

cm) and 14 (0.85 cm ± 0.33 cm) days old under control conditions compared to the WT

(Fig. 4E,F, 5C,G).

(40)

3.3.3 FMT-OE mutants have a lower rate of germination, take longer to germinate, and have shorter roots and leaves

Under control conditions, FMT-OE mutant plants had a much lower rate of germination, at only 82.33% ± 9.77% (Fig. 4A). These plants also took longer to germinate (3.12 days

±0.64 days) compared to WT, fis1A, and fmt plants (Fig. 4B). Despite this delayed germination, these mutants did not take longer to flower (29.83 days ±1.16 days) compared to the WT (Fig. 4D). Similar to fmt mutants, leaf length was significantly shorter in FMT-OE mutants (0.90 cm ±0.26 cm) versus the WT (Fig. 4C, 5P).

Interestingly, FMT-OE mutants displayed significantly shorter roots at 7 days old (0.21

cm ± 0.10 cm), but not at 14 days old (1.25 cm ±0.51 cm) under control conditions

compared to the WT (Fig. 4E,F, 5D, H).

(41)

Figure 4: Phenotypic differences between WT, fis1A, fmt, and FMT-OE lines under control

conditions. A) % Germination, B) Days to germination, C) Leaf length, D) Days to flowering, E) Root

length at 7 days old, F) Root length at 14 days old. Statistical analysis indicates significant differences

(****, P ≤ 0.0001, *** P ≤ 0.001, **, P ≤ 0.01, ns = not significant, P > 0.05) compared with controls using

one-way ANOVA.

(42)

Figure 5: Visualization of phenotypic differences between WT, fis1A fmt, and FMT-OE lines under

control and salt-stressed conditions. A-D) Arabidopsis seedlings at 7 days old under control conditions

on MS-Agar plates. A) WT, B) fis1A, C) fmt, D) FMT-OE. E-H) Arabidopsis seedlings at 14 days old under

control conditions on MS-Agar plates. E) WT, F) fis1A, G) fmt, H) FMT-OE. I-L) Arabidopsis seedlings at

14 days old under salt-stressed conditions on 125mM NaCl MS-Agar plates. I) WT, J) fis1A, K) fmt, L)

FMT-OE. M-P) Arabidopsis seedlings at 7 days old under control conditions in the soil. M) WT, N) fis1A,

O) fmt, P) FMT-OE. Scale bar = 1 cm.

(43)

3.4 Effects of salt stress on fis1A, fmt, and FMT-OE mutants and WT plants

In order to examine the role of the FIS1A and FMT mitochondrial proteins in response to salt stress, fis1A, fmt, and FMT-OE mutants were exposed to either 125mM NaCl stress on MS-Agar media, or gradual salt stress ranging from 50-140mM NaCl in the soil.

Additionally, to examine the effects of salt stress on WT Arabidopsis plants, and to serve as a control, these plants were also exposed to salt stress on plates and in the soil. On 125mM NaCl MS-Agar plates, WT plants did not have a significant difference in percent germination (Fig. 6A), but took longer to germinate compared to control conditions (3.64 days ±0.43 days compared to 1.24 days ±0.43 days, respectively) (Fig. 6B). These plants also had significantly shorter roots compared to WT plants under control conditions (0.99 cm ±0.33 cm compared to 1.56 cm ±0.48 cm, respectively) (Fig. 6E, 5I). Under salt- stressed conditions in the soil, WT leaves were significantly shorter compared to the WT control (0.90 cm ±0.15 cm compared to 1.23 cm ±0.25 cm, respectively) (Fig. 6C), and these plants took significantly longer to flower compared to WT plants under control conditions (31.37 days ±2.21 days compared to 28.89 days ±1.28 days, respectively) (Fig.

6D).

(44)

Figure 6: Salt stress affects days to germination and flowering, as well as leaf and root length in wild

type Arabidopsis. A) % Germination, B) Days to germination, C) Leaf length, D) Days to flowering, E)

Root length at 14 days old. Black bars indicate control conditions, grey bars indicate salt-stressed

conditions. Statistical analysis indicates significant differences (****, P ≤ 0.0001, ***, P ≤ 0.001)

compared with controls using two-tailed Student’s t test.

(45)

On 125mM NaCl MS-Agar plates, fis1A mutants germinated at relatively the same rate compared to control conditions (96.5% ±4.94% compared to 98.66% ±3.26%, respectively) (Fig. 7A), however these plants took longer to germinate compared to control conditions (3.84 days ±0.80 days compared to 1.5 days ±0.51 days, respectively) (Fig. 7B). These plants also had significantly shorter roots compared to fis1A plants under control conditions (0.64 cm ±0.25 cm compared to 0.85 cm ±0.43 cm, respectively) (Fig. 7E, 5J). Under salt stress conditions in the soil, fis1A leaves were significantly shorter compared to controls (0.82 cm ±0.13 cm compared to 1.03 cm ±0.21 cm, respectively) (Fig.7C), and these plants took significantly longer to flower compared to controls (34.31 days ±2.96 days compared to 29.4 days ±1.99 days, respectively) (Fig.

7D).

(46)

Figure 7: Salt stress affects days to germination and flowering, as well as leaf and root length in fis1A

mutants. A) % Germination, B) Days to germination, C) Leaf length, D) Days to flowering, E) Root length

at 14 days old. Black bars indicate control conditions, grey bars indicate salt-stressed conditions. Statistical

analysis indicates significant differences (****, P ≤ 0.0001, **, P ≤ 0.01) compared with controls using

two-tailed Student’s t test.

(47)

Compared to WT plants under salt-stressed conditions, salt-stressed fis1A mutant plants

did not have a significantly different germination rate (Fig. 8A), nor did they take longer

to germinate (Fig. 8B). These mutants also did not have significantly shorter roots at 14

days old compared to the WT under the same conditions (Fig. 8E). Under salt-stressed

conditions in the soil, the leaf length of fis1A mutants was not significantly shorter

compared to WT plants under the same conditions (Fig. 8C), although the number of days

to flowering was significantly increased (Fig. 8D).

(48)

Figure 8: Days to flowering is increased in fis1A mutants compared to the WT under salt-stressed conditions. A) % Germination, B) Days to germination, C) Leaf length, D) Days to flowering, E) Root length at 14 days old. Black bars indicate control conditions, grey bars indicate salt-stressed conditions.

Statistical analysis indicates significant differences (**, P ≤ 0.01, ns = not significant, P > 0.05) compared

with controls using two-tailed Student’s t test (A, B, C) or two-way ANOVA (C, E).

(49)

On 125mM NaCl MS-Agar plates, fmt mutants germinated at the same rate compared to control conditions (98.25% ±2.36% compared to 98.6% ±3.13%, respectively) (Fig. 9A), however these plants took much longer to germinate compared to controls (3.84 days

±0.98 days compared to 1.3 days ±0.48 days, respectively) (Fig. 9B). These plants did not have significantly shorter roots compared to fmt plants under control conditions (0.75 cm ±0.28 cm compared to 0.85 cm ±0.33 cm, respectively) (Fig. 9E, 7K). Under salt stress conditions in the soil, the leaves of fmt mutants were not significantly shorter compared to controls (0.83 cm ±0.16 cm compared to 0.93 cm ±0.20 cm, respectively) (Fig. 9C), nor did these plants take longer to flower compared to controls (33.42 days

±3.24 days compared to 33.57 days ±2.82 days, respectively) (Fig. 9D).

(50)

Figure 9: Salt stress affects days to germination in fmt mutants. A) % Germination, B) Days to

germination, C) Leaf length, D) Days to flowering, E) Root length at 14 days old. Black bars indicate

control conditions, grey bars indicate salt-stressed conditions. . Statistical analysis indicates significant

differences (****, P ≤ 0.0001) compared with controls using two-tailed Student’s t test.

(51)

Compared to WT plants under salt-stressed conditions, fmt mutant plants did not have a significantly different germination rate, nor did they take longer to germinate (Fig.

10A,B). These mutants also had shorter roots, although it was not statistically significant

(P= 0.0605) (Fig. 10E). However, under salt-stressed conditions in the soil, leaves of fmt

mutants were significantly shorter and days to flowering was significantly longer

compared to WT plants under the same conditions (Fig. 10C,D).

(52)

Figure 10: Days to flowering is increased and root and leaf length are decreased in fmt mutants

compared to the WT under salt-stressed conditions. A) % Germination, B) Days to germination, C)

Leaf length, D) Days to flowering, E) Root length at 14 days old. Black bars indicate control conditions,

grey bars indicate salt-stressed conditions. Statistical analysis indicates significant differences (*, P ≤ 0.05)

compared with controls using two-tailed Student’s t test (A, B) or two-way ANOVA (C, D, E).

Abbildung

Fig.   figure
Figure  1:  Mechanistic  action  of  tropic  growth  in  the  Arabidopsis  thaliana  root
Table 1: Antibiotics used in this study
Table 2: Primers used in this study
+7

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 The established sample preparation conditions could be successfully used for single particle electron microscopy of TIM22 and respiratory chain complexes, but not for

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Proteins destined for the mitochondrial matrix, or the inner membrane, are imported by the presequence translocase of the inner membrane (TIM23 complex).. The molecular

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The results confirmed the higher proportion of xylose in EPs-I than in EPs-II and indicate that this sugar occurs as single side chains linked to the mannan backbone and/or

The greater apoplastic protein content was the consequence of stimulated protein synthesis in the presence of NaCl, as evidenced by increased incorporation of [ 35 S]-methionine

But when expressed in terms of amount per leaf (µg), no difference was witnessed. The ambiguity in the amounts could be related to the limited development of leaf